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Regulation of CFTR by Protein Kinase C

蛋白激酶 C 对 CFTR 的调节

基本信息

批准号：
6758559
负责人：
CAROLE M LIEDTKE
金额：
$ 26.78万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-06-10 至 2006-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6758559
关键词：
antisense nucleic acid chloride channels cystic fibrosis enzyme activity enzyme inhibitors isozymes protein binding protein isoforms protein kinase C protein protein interaction respiratory epithelium tissue /cell culture

项目摘要

Cystic fibrosis is a disease of electrolyte transport abnormalities, which has, as its genetic basis, a mutation of the cystic fibrosis transmembrane regulator (CFTR), an apical secretory Cl channel. Correction of airway dysfunction in CF has centered on therapeutic approaches to activate apical Cl channels other than CFTR and genetic alteration of CFTR to correct abnormal Cl secretion and thus offset excess mucus accumulation. CFTR is regulated primarily by CAMP, however, the PI discovered that constitutive activity of PKC-epsilon is necessary for CAMP-dependent regulation of CFTR. Thus, regulation of CFTR function is more complicated than just cAMP-dependent phosphorylation of CFTR. New pilot studies indicate an association between PKC-epsilon and CFTR that might represent direct or indirect binding. In addition to CFTR, other tracheal epithelial proteins associate with PKC-epsilon, including scaffold proteins which might form a multiprotein complex that tethers PKC-epsilon proximal to its target. The hypothesis of this grant proposal is that PKC-epsilon regulates CFTR function through phosphorylation. This will be studied in detail in the following specific aims: 1) To determine whether activity of PKC- epsilon regulates its interaction with CFTR. Activity will be manipulated using PKC inhibitors or, to also decrease mass, with antisense oligonucleotides to PKC-epsilon. Enzyme activity will be correlated with co-purification of PKC-epsilon with CFTR and phosphorylation of wild type CFTR. Whether trafficking competent and incompetent mutant CFTR (G551D, deltaF508, respectively) alter CFTR interaction with PKC-epsilon and its phosphorylation of CFTR will be determined. 2) To determine whether activity of PKC-epsilon is regulated by association with a multiprotein complex. Binding of PKC-epsilon with recombinant or endogenous proteins (CFTR, RACK, actin) will be measured by direct binding or overlay assay and quantitated as a K-m for binding. Binding of activated and/or inactive enzyme and specificity for PKC isotype will be determined. Activity will be manipulated by omitting PKC activators or adding PKC inhibitor and by downregulating binding protein using an antisense approach. Whether rapid loss of CFTR function by PKC inhibitor is correlated to activity of serine/threonine protein phosphatase(s) and its association with PKC-epsilon finding partners will be determined. 3) To identify site(s) of interaction between PKC- epsilon and target/binding protein. Sequence motifs in specific domains of PKC-epsilon and/or binding will be predicted and tested using peptides to inhibit binding and cAMP-dependent activation of CFTR.

囊性纤维化是一种电解质转运异常的疾病，其遗传基础是囊性纤维化跨膜调节因子（CFTR）（顶端分泌氯离子通道）的突变。 CF 气道功能障碍的纠正主要集中在激活除 CFTR 之外的心尖 Cl 通道的治疗方法和 CFTR 的基因改变以纠正异常的 Cl 分泌，从而抵消过量的粘液积聚。 CFTR 主要受 CAMP 调节，然而，PI 发现 PKC-epsilon 的组成型活性对于 CFTR 的 CAMP 依赖性调节是必需的。因此，CFTR 功能的调节比 CFTR 的 cAMP 依赖性磷酸化更为复杂。新的试点研究表明 PKC-epsilon 和 CFTR 之间的关联可能代表直接或间接结合。除了 CFTR 之外，其他气管上皮蛋白也与 PKC-epsilon 相关，包括可能形成多蛋白复合物的支架蛋白，将 PKC-epsilon 束缚在其靶标附近。该资助提案的假设是 PKC-epsilon 通过磷酸化调节 CFTR 功能。这将在以下具体目标中进行详细研究： 1) 确定 PKC-ε 的活性是否调节其与 CFTR 的相互作用。将使用 PKC 抑制剂来控制活性，或者为了减少质量，使用 PKC-ε 的反义寡核苷酸。酶活性与 PKC-ε 与 CFTR 的共纯化以及野生型 CFTR 的磷酸化相关。将确定有能力和无能力的突变体 CFTR（分别为 G551D、deltaF508）是否会改变 CFTR 与 PKC-epsilon 的相互作用及其 CFTR 的磷酸化。 2)确定PKC-ε的活性是否通过与多蛋白复合物的结合来调节。 PKC-ε 与重组或内源蛋白（CFTR、RACK、肌动蛋白）的结合将通过直接结合或重叠测定进行测量，并以结合的 K-m 进行定量。将确定活化和/或失活酶的结合以及PKC同种型的特异性。通过省略 PKC 激活剂或添加 PKC 抑制剂以及使用反义方法下调结合蛋白来操纵活性。将确定 PKC 抑制剂引起的 CFTR 功能的快速丧失是否与丝氨酸/苏氨酸蛋白磷酸酶的活性相关及其与 PKC-ε 寻找伴侣的关系。 3) 鉴定 PKC-ε 和靶/结合蛋白之间相互作用的位点。将使用肽来预测和测试 PKC-ε 和/或结合的特定结构域中的序列基序，以抑制 CFTR 的结合和 cAMP 依赖性激活。